Method for kidney disease detection and treatment

ABSTRACT

A method is disclosed for diagnosing early stage of a disease in which an intact protein found in urine is an indicator of the disease. The method includes assaying urine sample to detect the presence of modified protein using either immunological or non-immunological technique. Methods for preventing and treating the disease are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation-in-Part of and claims priority toparent application Ser. No. 09/415,217, filed Oct. 12, 1999.

FIELD OF THE INVENTION

The present invention relates to methods of detecting an early stage ofrenal disease and/or renal complications of a disease. The inventionalso relates to preventing and treating the disease.

BACKGROUND OF THE INVENTION

The appearance of excess protein such as albumin in the urine isindicative of kidney disease. Diabetic nephropathy is such a disease. Bythe time the excess albumin is detected, kidney disease has progressed,possibly to a stage where it is irreversible and treatment has littleeffect. Therefore it is an object of the invention to provide a testthat is more sensitive than the currently known radioimmunoassay todetect such a disease as early as possible so that the disease can beeither prevented or a treatment protocol commenced early on in thedisease.

Specific proteinuria, and in particular, albuminuria (micro- andmacro-), is a marker of disease including renal disease(glomerulonephritis, bacterial and viral glomerulonephritides, IgAnephropathy and Henoch-Schönlein Purpura, membranoproliferativeglomerulonephritis, membranous nephropathy, Sjögren's syndrome, diabeticnephropathy, nephrotic syndrome (minimal change disease, focalglomerulosclerosis and related disorders), acute renal failure, acutetubulointerstitial nephritis, pyelonephritis, GU tract inflammatorydisease, Preclampsia, renal graft rejection, leprosy, refluxnephropathy, nephrolithiasis), genetic renal disease (medullary cystic,medullar sponge, polycystic kidney disease (autosomal dominantpolycystic kidney disease, autosomal recessive polycystic kidneydisease, tuborous sclerosis), von Hippel-Lindau disease, familialthin-glomerular basement membrane disease, collagen III glomerulopathy,fibronectin glomerulopathy, Alport's syndrome, Fabry's disease,Nail-Patella Syndrome, congenital urologic anomalies), monoclonalgammopathies (multiple myeloma, amyloidosis and related disrders),febrile illness (familial Mediterannean fever, HIV infection—AIDS),inflammatory disease (systemic vasculitides (polyarteritis nodosa,Wegener's granulomatosis, polyarteritis, necrotizing and crescenticglomerulonephritis), polymyositisdermatomyositis, pancreatitis,rheumatoid arthritis, systemic lupus erythematosus, gout), blooddisorders (sickle cell disease, thrombotic thrombocytopenia purpura,hemolytic-uremic syndrome, acute corticol necrosis, renalthromboembolism), trauma and surgery (extensive injury, bums, abdominaland vascular surgery, induction of anesthesia), drugs (penicillamine,steroids) and drug abuse, malignant disease (epithelial (lung, breast),adenocarcinoma (renal), melanoma, lymphoreticular, multiple myeloma),circulatory disease (myocardial infarction, cardiac failure, peripheralvascular disease, hypertension, coronary heart disease,non-atherosclerotic cardiovascular disease, atheroscleroticcardiovascular disease), skin disease (psoriasis, systemic sclerosis),respiratory disease (COPD, obstructive sleep apnoea, hypoia at highaltitude) and endocrine disease (acromegaly, diabetes mellitus, anddiabetes insipidus).

Kidney disease may result from bacterial infection, allergies,congenital defects, stones, antibiotics, immunosuppressives,antineoplastics, nonsteroidal anti-inflammatory drugs, analgesics, heavymetals, tumors, chemicals.

The applicant has found that proteins, including albumin, are normallyexcreted as a mixture of native protein and fragments that arespecifically produced during renal passage (Osicka, T. M. et al. (1996)Nephrology, 2, 199-212). Proteins are heavily degraded during renalpassage by post-glomerular (basement membrane) cells, which may includetubular cells. Lysosomes in renal tubular cells may be responsible forthe breakdown of proteins excreted during renal passage (see FIG. 1).The breakdown products are excreted into the tubular lumen. In normalindividuals, most of the albumin in the urine is fragmented.

When lysosome activity or intracellular processes directing substratesto lysosomes is reduced, more of the high molecular weight, andsubstantially full length albumin appears in the urine. This reflects animbalance in the cellular processes in the kidney tissue.

Until now, it was thought that conventional radioimmunoassay wassuitable for detecting all (total) of a specific protein in a sample.But the total content of the protein may include more than those thatare identifiable by known antibodies using conventional radioimmunoassay(RIA). Currently available radioimmunoassays rely on antibodies todetect proteins such as albumin. Antibody detection is very accuratedown to nanogram levels. However, the specificity of the antibodiesinfluences detection of the protein. The antibody detects certainepitopes. If the specific epitope on the albumin is absent, altered ormasked, or the albumin is modified in any other way so that the antibodyfails to detect the albumin, conventional radioimmunoassays may notprovide a true representation of the true amount of albumin present in aurine sample.

Methods of detecting early signs of a disease, including kidney disease,determining a patient's propensity for the disease, preventing the onsetof the disease, and treating the disease at the earliest stage possible,as well as a method for determining the total content of a specificprotein in a sample, are some of the objects of the invention.

SUMMARY OF THE INVENTION

The present invention is directed to a method of diagnosing early stageof renal disease and/or renal complications of a disease, comprising:

(a) separating all of the proteins in a urine sample; and

(b) detecting a modified form of a protein in the sample, whereindetection of the modified protein is indicative of an early stage of therenal disease and/or renal complications of a disease.

Although not limited to any particular disease, according to the methodof the invention, the disease sought to be diagnosed includesnephropathy, diabetes insipidus, diabetes type I, diabetes II, renaldisease (glomerulonephritis, bacterial and viral glomerulonephritides,IgA nephropathy and Henoch-Schönlein Purpura, membranoproliferativeglomerulonephritis, membranous nephropathy, Sjögren's syndrome,nephrotic syndrome (minimal change disease, focal glomerulosclerosis andrelated disorders), acute renal failure, acute tubulointerstitialnephritis, pyelonephritis, GU tract inflammatory disease, Pre-clampsia,renal graft rejection, leprosy, reflux nephropathy, nephrolithiasis),genetic renal disease (medullary cystic, medullar sponge, polycystickidney disease (autosomal dominant polycystic kidney disease, autosomalrecessive polycystic kidney disease, tuborous sclerosis), vonHippel-Lindau disease, familial thin-glomerular basement membranedisease, collagen III glomerulopathy, fibronectin glomerulopathy,Alport's syndrome, Fabry's disease, Nail-Patella Syndrome, congenitalurologic anomalies), monoclonal gammopathies (multiple myeloma,amyloidosis and related disorders), febrile illness (familialMediterranean fever, HIV infection—AIDS), inflammatory disease (systemicvasculitides (polyarteritis nodosa, Wegener's granulomatosis,polyarteritis, necrotizing and crescentic glomerulonephritis),polymyositis-dermatomyositis, pancreatitis, rheumatoid arthritis,systemic lupus erythematosus, gout), blood disorders (sickle celldisease, thrombotic thrombocytopenia purpura, hemolytic-uremic syndrome,acute cortical necrosis, renal thromboembolism), trauma and surgery(extensive injury, bums, abdominal and vascular surgery, induction ofanesthesia), drugs (penicillamine, steroids) and drug abuse, malignantdisease (epithelial (lung, breast), adenocarcinoma (renal), melanoma,lymphoreticular, multiple myeloma), circulatory disease (myocardialinfarction, cardiac failure, peripheral vascular disease, hypertension,coronary heart disease, non-atherosclerotic cardiovascular disease,atherosclerotic cardiovascular disease), skin disease (psoriasis,systemic sclerosis), respiratory disease (COPD, obstructive sleepapnoea, hypoia at high altitude) and endocrine disease (acromegaly,diabetes mellitus, diabetes insipidus).

In addition, the method can be practiced using any protein, preferably,albumin, globulin (α-globulin(α₁-globulin,α₂-globulin),β-globulin,γ-globulin), euglobulin, pseudoglobulin I andII, fibrinogen, α₁ acid glycoprotein (orosomucoid), α₁ glycoprotein, α₁lipoprotein, ceruloplasmin, α₂ 19 S glycoprotein, β₁ transferrin, β₁lipoprotein, immunoglobulins A, E, G, and M, horseradish peroxidase,lactate dehydrogenase, glucose oxidase, myoglobin, lysozyme, proteinhormone, growth hormone, insulin, or parathyroid hormone.

The method can be practiced using non-antibody means, using such methodsas chromatography, electrophoresis, or sedimentation, which furtherinclude such methods as partition chromatography, adsorptionchromatography, paper chromatography, thin-layer chromatography,gas-liquid chromatography, gel chromatography, ion-exchangechromatography, affinity chromatography, or hydrophobic interactionchromatography, moving-boundary electrophoresis, zone electrophoresis,or isoelectric focusing. Albumin detection with specific albumin dyesmay also be used.

In particular, the method of the invention is directed to using ahydrophobic interaction chromatography in a high-pressure liquidchromatography (HPLC) apparatus.

The present invention is also directed to an antibody detecting methodfor diagnosing early stage of renal disease and/or renal complicationsof a disease. The method of the invention is accomplished by assayingfor an intact/modified protein that is not normally identifiable inurine using conventional means. The intact/modified protein of theinvention is present in the urine sample of a diseased person or aperson who is predisposed to a disease before the native protein can bedetected. Therefore, the detection of an intact/modified protein in aurine sample indicates at an early stage that the subject is eitherdiseased or predisposed to the disease, even though the subject mayotherwise appear to be normal. An assay method of the invention includesdetecting an intact/modified protein by an antibody specific for boththe modified and unmodified forms of the protein. Preferably, theantibody is specific for the modified protein. The antibody can beattached to an enzymatic, radioactive, fluorescent or chemiluminescentlabel, wherein the detecting step comprises radioimmunoassay,immunoradiometric assay, fluorescent immunoassay, enzyme linkedimmunoassay, or protein A immunoassay.

In the method of the invention, the early stage of the disease isdiagnosed when the modified protein is present in the urine inincreasing amounts over time, and conventional radioimmunoassay does notdetect the modified protein.

The present invention is also directed to an article of manufacture fordiagnosing an early stage of renal disease and/or renal complications ofa disease, comprising:

(a) a container comprising a labeled antibody specific for a modifiedform of the protein;

(b) a container comprising reagents for developing antibody reaction;and

(c) instructions on how to use components (a) and (b) to carry out thediagnosis.

In addition, the present invention is also directed to a method fordetermining a treatment agent for renal disease and/or renalcomplications of a disease, comprising:

(a) administering to a person in need thereof an agent that is suspectedof being able to treat the disease;

(b) obtaining a urine sample from the person; and

(c) assaying for a modified form of the protein in the sample, whereineither presence or lack of presence of the modified form of the proteinin the urine or decreasing amount of the modified form of the proteinover time indicates that the agent is a treatment agent for the renaldisease and/or renal complications of a disease.

The invention is also directed to a method for treating a personsuffering from a disease in which a diseased state is indicated byspecific proteinuria, comprising administering a therapeuticallyeffective amount of the treatment agent obtained according to the abovemethod to a person in need thereof. Preferably, the treatment agent is alysosome activating compound.

Another object of the invention is to determine the sum of modified andunmodified forms of a of a specific protein in a sample, comprising:

(a) separating all of the proteins in the sample;

(b) detecting modified and unmodified forms of the specific protein; and

(c) integrating the modified and unmodified forms of the specificprotein to determine the sum of the specific protein in the sample.

Preferably, the sample is a biological sample, such as urine.

These and other objects of the invention will be more fully understoodfrom the following description of the invention, the referenced drawingsattached hereto and the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the progress of filtered intact albumin into tubularcells and breakdown of albumin to provide excreted albumin fragments.

FIGS. 2a and 2 b illustrate a representative profile of (³H) HSA in (a)urine and (b) plasma collected from normal, healthy volunteers by sizeexclusion chromatography. Urine contains mostly fragmented albumin. Andplasma contains mostly intact albumin.

FIG. 3 illustrates urine from normal, healthy volunteer showing afragmented albumin peak, but no intact albumin peak from size exclusionchromatography.

FIG. 4 illustrates urine from a diabetic patient showing both intact andfragmented albumin peaks from size exclusion chromatography.

FIG. 5 illustrates a HPLC profile of albumin alone.

FIG. 6 illustrates the HPLC profile of plasma from normal, healthyvolunteer showing albumin peaks.

FIG. 7 shows the HPLC profile of urine from normal, healthy volunteerwith fragmented products of albumin but no intact albumin peak.

FIG. 8 shows the HPLC profile of a urine sample from a normoalbuminuricdiabetic patient showing albumin breakdown products and a small-modifiedalbumin peak at approximately 39-44 minutes retention time.

FIG. 9 shows the HPLC profile of urine from a normoalbuminuric diabeticpatient showing signs of kidney failure and the presence of thecharacteristic spiked albumin peak at approximately 39-44 minutesretention time.

FIG. 10 illustrates a HPLC profile of a normoalbuminuric diabeticpatient showing signs of kidney failure and the presence of thecharacteristic spiked modified albumin peak at approximately 39-44minutes retention time.

FIG. 11 illustrates a HPLC of a macroalbuminuric diabetic patientshowing high levels of the normal albumin as well as the characteristicspiked appearance at approximately 39-44 minutes retention time.

FIG. 12 illustrates a longitudinal study of a patient in which themodified protein was detected at a time prior to onset of diabeticnephropathy, indicating predisposition to diabetic nephropathy, and thedelay in treatment caused by relying on conventional RIA methods.

FIG. 13 illustrates a longitudinal study of a patient in which themodified protein was detected at a time prior to onset of diabeticnephropathy, indicating predisposition to diabetic nephropathy, and thedelay in treatment caused by relying on conventional RIA methods.

FIG. 14 illustrates a longitudinal study of a patient in which themodified protein was detected at a time prior to onset of diabeticnephropathy, indicating predisposition to diabetic nephropathy, and thedelay in treatment caused by relying on conventional RIA methods.

FIG. 15 shows the HPLC chromatogram used as a criterion of purity of themodified albumin of Example 4.

DETAILED DESCRIPTION OF THE INVENTION

The applicant has discovered that when proteins, including major plasmaproteins such as albumin and immunoglobulin, are filtered by the kidneythey are subsequently degraded by cells in the kidney prior to thematerial being excreted. It is likely that filtered proteins are takenup by tubular cells. Tubular cells lie beyond the kidney filter and comein direct contact with the primary filtrate. When proteins areinternalized by the tubular cells, they are directed towards thelysosomes, where they are partially degraded to various size fragments,and then regurgitated to outside the cell. These regurgitated fragments,of which there may be at least 60 different fragments generated from anyone particular type of protein, are then excreted into the urine.

The applicant has discovered that in renal disease fragmentation ofproteins is inhibited. This means that substantially full-lengthfiltered proteins will be excreted in a person suffering from renaldisease. This transition from fragmentation to inhibition offragmentation of excreted proteins is a basis for the development of newdrugs and diagnostic assays. For example, initial changes that occurwith the onset of renal complications in diabetes are associated with achange in the fragmentation profile of excreted albumin. This leads toan apparent microalbuminuria, which is synonymous with the developmentof diabetic nephropathy. It is likely that this is due to an inhibitionin the lysosomal activity of tubular cells in diabetes. Thus, drugs canbe formulated to turn on lysosomal activity in diabetes where renalcomplications are occurring. The drugs may also be useful in other renaldiseases where lysosomal activities are affected, or in diabetes withoutrenal complications in situations where lysosomal activity is turned offin non renal tissues. Such drugs include antiproliferative drugs, suchas anti cancer drugs or antibodies to neutralize TGF beta.

The applicant has discovered a unique assay for detecting modified formsof specific proteins, which are detected in the urine of certainsubjects before the unmodified form of the specific protein is detectedusing conventional assays, such as radioimmunoassays. Detection of themodified protein is predictive of a predisposition to renal disease.

Definitions

“Fragmented protein or fragment albumin” includes post-glomerularbreakdown products after chemical, enzymatic or physical breakdown thatoccurs during renal passage. These components have a reduced size and/ormay have changed hydrophobicity.

“Intact albumin, modified albumin, or modified form of albumin” as usedherein means a compound having similar size and structuralcharacteristics to native albumin, wherein the amino acid sequence issubstantially the same as the native albumin. It is preferably afiltered intact protein. It elutes at or near the same position asnative albumin on high-pressure liquid chromatography (HPLC) (FIG. 5).However, the structure has been modified biochemically either by minorenzyme mediated modification or addition to its basic structure and/orphysically through a change in its three dimensional structure so thatit escapes detection by conventionally used anti-albumin antibodies.Biochemical modification may be made by enzymes such as endo- orexo-peptidases. The 3D structure of albumin may have been altered insome way. Ligands may have bound to the albumin, or it may be anycombination of these. The modified albumin detected in the method of theinvention is not detectable by current and conventionalradioimmunoassays using available antibodies and is not a fragment.

Conventional anti-albumin antibodies can be purchased from any purveyorof immunochemicals. For example, monoclonal antibody catalog numbersA6684 (clone no. HSA-11), and A2672 (clone no. HSA-9), as well as liquidwhole serum, lyophilized fractionates, liquid IgG fraction, and themonoclonal antibodies in liquid ascites fluids form, can be obtainedfrom Sigma, St. Louis, Mo., as found in the Immunochemicals section atpages 1151-1152 in the 1994 Sigma—Biochemicals Organic Compounds forResearch and Diagnostic Reagents catalog.

As used herein, intact/modified albumin includes albumin that issubstantially full-length, fragmented, chemically modified, orphysically modified. As used herein, intact/modified albumin is meant toindicate albumin that is less than, equal to, or greater in molecularweight than the full-length albumin, and elutes at or near the nativealbumin position in a separation medium, such as chromatography,preferably HPLC, and most preferably hydrophobicity HPLC. As usedherein, fragmented albumin is meant to refer to the fragment of albuminthat is not detected by conventional anti-albumin antibody, and itspresence is detected in diagnosing an early stage of renal diseaseand/or renal complications of a disease. The detection of the presenceof intact/modified albumin is an indication of a predisposition to renaldisease.

“Intact protein, modified protein or modified form of a protein” as usedherein includes those forms of substantially full-length protein whichare undetectable by conventional radioimmunoassay. The protein includes,but is not limited to, albumins, globulins (α-globulin(α₁-globulin,α₂-globulin),β-globulins,γ-globulins), euglobulins, pseudoglobulin I andII, fibrinogen, α₁ acid glycoprotein (orosomucoid), α₁ glycoprotein, α₁lipoproteins, ceruloplasmin, α₂ 19 S glycoprotein, β₁ transferrin, β₁lipoprotein, immunoglobulins A, E, G, and M, protein hormones includinggrowth hormone, insulin, parathyroid hormone and other proteinsincluding horseradish peroxidase, lactate dehydrogenase, glucoseoxidase, myoglobin, and lysozyme.

“Kidney disease” as used herein includes any malfunction of the kidney.Kidney disease may be identified by the presence of intact or modifiedalbumin in the urine. Preferably, an early diagnosis of the kidneydisease may be made by detecting the presence of modified protein in theurine, or an increase in the modified protein in the urine over time.

“Low lysosome activity” as used herein is compared against normal levelsof lysosome activity and/or lysosome machinery that traffics protein tothe lysosome in a normal individual. The activity is insufficient forthe lysosome to fragment proteins so that intact protein is excreted ata greater amount than at normally low levels.

“Lysosome-activating compound” as used herein refers to a compound thatis beneficial to reactivation of the lysosome. The compound may workdirectly or indirectly on the lysosome resulting in activation oflysosomal function. These compounds may be selected from the groupincluding, but not limited to, anticancer compounds, antiproliferationcompounds, paracetamol, vitamin A (retinoic acid) or derivatives ofretinol, or compounds, including antibodies, to neutralize TGF beta.

“Macroalbuminuria” is a condition where an individual excretes greaterthan 200 μg albumin/min in the urine as measured by conventionalradioimmunoassay (RIA).

“Microalbuminuria” is a condition where an individual excretes at least20 μg albumin/min in the urine as measured by conventionalradioimmunoassay (RIA). RIA measures down to 15.6 ng/ml and is able tomeasure albumin in urine of normal subjects who have clearance of lessthan 6 μg/min. However, when albumin excretion exceeds 20 μg/min,treatment of the kidney disease is limited and full recovery isdifficult from this point.

“Microalbuminuric” as used herein is a condition when albumin isdetected in the urine at an excretion rate of at least 20 μg/min asmeasured by conventional RIA.

As used herein, “native” and “unmodified” are used interchangeably todescribe a protein that is naturally found in an organism, preferably ahuman, which has not been modified by the filtering process of the renalglomeruli.

“Normal individual” as used herein is an individual who does not have adisease in which intact protein found in urine is an indicator of thedisease. Preferably, the disease is kidney disease.

“Normal levels of lysosome activity” are levels of lysosome activityfound in undiseased kidney of a normal individual.

“Normoalbuminuric” as used herein means a condition where albumin isexcreted in the urine and is not detectable by RIA, or less than 20μg/min (as measured by RIA) is excreted.

“Propensity for a disease” as used herein means that a disease mayresult in an individual as judged by a determination of the presence andexcretion rate of a modified protein such as modified albumin.

“Proteinuria” as used herein is the existence of protein in the urine,usually in the form of albumin, a protein that is soluble in water andcan be coagulated by heat. Related to this, “specific proteinuria”refers to the existence of a particular protein in the urine.

“Radioimmunoassay” as used herein is a method for detection andmeasurement of substances using radioactively labeled specificantibodies or antigens.

“Reactivation of the lysosome” as used herein includes an activation oflysosome activity preferably so that breakdown of proteins, particularlyalbumin, is increased compared with an inactivated state of thelysosome.

“Restore” as used herein means to restore in full or in part so that thecomponent being restored has an improved function compared with itsprevious function.

The “sum of intact and intact modified protein” as used herein refers tothe total amount of intact protein, and intact modified protein presentin a biological sample.

“Total protein” as used herein refers to a particular filtered proteinpresent in native, unmodified, modified or fragmented form that isexcreted in urine. It includes protein that is not detected byconventional radioimmunoassay or conventional methods, which arecurrently available to detect the protein. Preferably the protein isalbumin.

According to the present invention, the diseases to be treated include,but are not limited to renal disease (glomerulonephritis, bacterial andviral glomerulonephritides, IgA nephropathy and Henoch-SchönleinPurpura, membranoproliferative glomerulonephritis, membranousnephropathy, Sjögren's syndrome, diabetic nephropathy, nephroticsyndrome (minimal change disease, focal glomerulosclerosis, and relateddisorders), acute renal failure, acute tubulointerstitial nephritis,pyelonephritis, GU tract inflammatory disease, Pre-clampsia, renal graftrejection, leprosy, reflux nephropathy, nephrolithiasis), genetic renaldisease (medullary cystic, medullar sponge, polycystic kidney disease(autosomal dominant polycystic kidney disease, autosomal recessivepolycystic kidney disease, tuborous sclerosis), von Hippel-Lindaudisease, familial thin-glomerular basement membrane disease, collagenIII glomerulopathy, fibronectin glomerulopathy, Alport's syndrome,Fabry's disease, Nail-Patella Syndrome, congenital urologic anomalies)

In one aspect of the invention, there is provided a method fordetermining a propensity for or early diagnosis of renal disease and/orrenal complications of a disease. The method includes determining achange in the albumin content in a urine sample. The disease may be akidney disease, although not necessarily limited to a kidney disease.

In the method of the invention, albumin is used herein only as anexample of a protein to be detected in urine. When the albumin in apatient is analyzed by conventional RIA, it is expected that anormoalbuminuric patient or normal individual would have albumin in theurine in the range of 3-10 μg/min in young people and greater in olderpeople. However, normoalbuminuric patients also show levels of albuminin the urine if measured by HPLC. Applicant has found that these levelsmay be in the order of 5 μg/min. As kidney disease progresses, the levelof intact/modified albumin will increase to microalbuminuria levels inthe order of 20 to 200 μg/min as determined by RIA. This will be muchhigher when determined by HPLC or a method that determines the sum ofintact albumin and intact modified albumin. By monitoring the increasein intact/modified albumin, early signs of kidney disease may bedetected. However, these levels are not detectable by the methodscurrently available such as radioimmunoassay using antibodies currentlycommercially in use, possibly for the reason that antibodies detectcertain epitopes. If the albumin is modified in any way as describedabove, the epitope may be destroyed thereby leaving the modified albuminundetectable.

A patient suspected of having diabetic kidney disease will not showsigns of kidney degeneration until well after 10 to 15 years whenalbumin is detected by currently available methods such as RIA methods.Urinary excretion rates of at least 20 μg/min may be detected by RIAwhen an individual enters a microalbuminuric state. Again, by observingthe excretion of modified albumin, a change in the kidney and possiblyonset of a kidney disease may be detected.

A normoalbuminuric subject, or normoalbuminuric diabetic patient maycontinue to have a low albumin excretion rate of less than 20 μg/min asdetermined by RIA, for many years. The presence of albumin in the urineis a sign that functions of the kidney may be impaired. Once this levelbegins to change, treatment may be initiated.

In a normal individual a small amount of albumin is detectable in theurine. Total filtered albumin appears mainly as fragmented albumin inurine. Some albumin may be detected in normoalbuminuric individuals.However, the excretion rate of albumin in urine in a normoalbuminuricindividual may be as low as 5 μg/min. This level is generally detectableby RIA.

The modified protein of the invention can be detected by a variety ofmethods that are well-known in the art, including, but not limited tochromatography, electrophoresis and sedimentation, or a combination ofthese, which are described in Karger B L, Hancock W S (eds.) HighResolution Separation and Analysis of biological Macromolecules. Part AFundamentals in Methods in Enzymology, Vol. 270, 1996, Academic Press,San Diego, Calif., USA; Karger B L, Hancock W S (eds.) High ResolutionSeparation and Analysis of biological Macromolecules. Part BApplications in Methods in Enzymology, Vol. 271, 1996, Academic Press,San Diego, Calif., USA; or Harding S E, Rowe, A J, Horton J C (eds.)Analytical Ultracentrifugation in Biochemistry and Polymer Science.1992, Royal Soc. Chemistry, Cambridge, UK, which references areincorporated herein by reference in their entirety.

The electrophoresis method includes, but is not limited to,moving-boundary electrophoresis, zone electrophoresis, and isoelectricfocusing.

The chromatography method includes, but is not limited to, partitionchromatography, adsorption chromatography, paper chromatography,thin-layer chromatography, gas-liquid chromatography, gelchromatography, ion-exchange chromatography, affinity chromatography,and hydrophobic interaction chromatography. Preferably, the method is asizing gel chromatography and hydrophobic interaction chromatography.More preferably, the method is hydrophobic interaction chromatographyusing a HPLC column.

The modified protein can also be detected by the use of specific albumindyes. Such methods are described for example by Pegoraro, et al.American Journal of Kidney Diseases, Vo. 35, No. 4, April, 2000, pp.739-744, the entire disclosure of which is hereby incorporated byreference. The modified albumin, as well as whole albumin, aredetectable by this dye method to provide the sum of modified albumin andwhole or intact albumin. This detection method may be used with orwithout an initial separation of the albumin components form urine. Suchdyes normally do not detect fragments <10,000 in molecular weight, butwill detect the modified albumin.

In this dye method of detection, a dye such as Albumin Blue 580, isused. Such dyes are naturally non-fluorescent but fluoresce on bindingto intact albumin as well as the modified albumin, but do not bind toglobulins. Therefore, globulins do not interfere with the assay so thatmeasurements can be made in unfractionated urine.

Applicant has found that among diabetics, a normoalbuminuric diabeticpatient has almost undetectable levels of modified or fragments ofalbumin when analyzed by conventional RIA. They appear to be normal.However, when the urine is tested by HPLC, the levels of modifiedalbumin are much greater than found in a normal individual. Thisdifference in albumin may be attributed to the inability of conventionalRIA's to adequately detect all albumin (total albumin) in intact ormodified forms. Thus, HPLC is preferred for generating a fragmentationprofile. A fragmentation profile on HPLC is characterized by a series ofpeaks representing a number of species of albumin as fragments or inintact or modified forms.

In a preferred aspect of the present invention, the method ofdetermining a propensity for or early diagnosis of a kidney disease in asubject is determined before the subject becomes microalbuminuric.

Measuring albumin content in a sample by an HPLC method of the presentinvention may provide different results from its measurement byconventional RIA. In the HPLC technique, a low level of albumin isobserved in normal individuals. When the level of modified albuminbegins to be detected and its level increases, and progresses towardmicroalbuminuria then a patient can be determined to have a propensityfor kidney disease.

In a normal individual, the HPLC generated fragmentation profile ischaracterized by the absence of a peak in a region where full-lengthnative albumin elutes. Instead, multiple fragmented albumin isdetectable. A pure protein product (unmodified) produces essentially asingle peak. For example, using a hydrophobicity HPLC, albumin wasobserved to elute in the range of 39-44 minutes (FIG. 5). Thus, a normalindividual would provide a distinct fragmentation profile indicative ofan absence of kidney disease or no propensity for a kidney disease.However, as kidney disease progresses, an increasing amount of modifiedalbumin first, and then native form later are detectable. Thefragmentation profile begins to change and more products in the regionof full-length albumin manifests as additional spikes or an enlargedpeak indicative of more intact/modified albumin in the urine.

In a HPLC generated fragmentation profile of a urine sample, themodified albumin may appear in a region where native albumin elutes butmay be manifest as multiple peaks indicating the presence of multipleforms of modified albumin.

In a further preferred embodiment, the propensity for kidney disease maybe measured by determining the presence of or identifying at least onespecies of modified albumin. This may be determined or identified by thepresence of a specific peak on a HPLC profile, preferably the peak iswithin the range of position that corresponds to the elution position ofthe native albumin.

A HPLC column for detecting modified albumin or unmodified albumin maybe a hydrophobicity column, such as Zorbax 300 SB-CB (4.6 mm×150 mm). A50 μl sample loop may be used. Elution solvents suitable for HPLC indetecting albumin and its breakdown products may include standardelution solvents such as acetonitrile solvents. Preferably a buffer ofwater/1% trifluoro acetic acid (TFA) followed by a buffer of 60%acetonitrile/0.09% TFA may be used. A gradient of 0 to 100% of a 60%acetonitrile/0.09% TFA has been found to be suitable.

Suitable HPLC conditions for a hydrophobicity column may be as follows:

Solvent A H₂O, 1% trifluoro acetic acid

Solvent B 60% acetonitrile, 0.09% TFA

Solvent A2 99.96>00.00:49.58 min

Pressure 9.014Mpascalls (˜1100 psi)

Solvent B2 0.04>100.0:49.58 min

Pressure 7.154Mpascalls

The wavelength used in HPLC may be approximately 214 nm.

Modified albumin may elute between 39-44 minutes (FIG. 5). Albuminfragments may elute much earlier, mainly at less than 20 minutes.

The method for determining the propensity for kidney disease isapplicable to any individual. Kidney disease may be caused by a numberof factors including bacterial infection, allergic, congenital defects,stones, tumors, chemicals or from diabetes. Preferably, the method isapplicable for determining a propensity for kidney disease in diabeticpatients that may progress to a kidney disease. Preferably, theindividual is a normoalbuminuric diabetic. However, normal individualsmay be monitored for propensity for the disease by determining increasedlevels of intact or modified albumin in the urine.

The method of the invention can be carried out using non-antibodyseparation procedures as described above. However, antibody specific formodified protein may also be used to detect the presence of the modifiedprotein.

The antibody to the modified protein may be obtained using the followingmethod. The procedure is described specifically for albumin by way ofexample only, and can be readily applied to antibody production againstany other protein in the urine. The method seeks to determine whichmodified albumin molecule is the most sensitive marker to identifydiabetic patients, for example, who will progress to kidneycomplications.

The modified albumin is characterized by carrying out a quantitativeseparation of the modified albumin molecules, such as by preparativeHPLC. The modified proteins are analyzed for ligand binding, such asglycation. Subsequently, amino acid sequence of the individual modifiedprotein is determined, preferably by mass spectrometry using methodsdescribed in Karger B L, Hancock WS (eds.) High Resolution Separationand Analysis of biological Macromolecules. Part A Fundamentals inMethods in Enzymology, Vol. 270, 1996, Academic Press, San Diego,Calif., USA; or Karger B L, Hancock W S (eds.) High ResolutionSeparation and Analysis of biological Macromolecules. Part BApplications in Methods in Enzymology, Vol. 271, 1996, Academic Press,San Diego, Calif., USA, for example, which references are incorporatedherein by reference in their entirety. In a preferred embodiment, theremay be about 3 to 4 modified albumin species.

The method of generating antibody against the modified albumin seeks todevelop a diagnostic immunoassay for the modified albumin that predictsthose diabetic patients, for example, that progress to kidneycomplications. To accomplish this, sufficient quantities of modifiedalbumin is prepared by HPLC. Antibodies are made by sequential injectionof the modified albumin in an animal such as a rabbit, to generate goodtiter, and the antibodies are isolated using conventional techniquesusing methods described in Goding J W, Monoclonal Antibodies: Principlesand Practice. Production and Application of Monoclonal Antibodies inCell Biology, Biochemistry and Immunology, 2nd Edition 1986, AcademicPress, London, UK; or Johnstone A, Thorpe R, Immunochemistry inPractice, 3rd edition 1996, Blackwell Science Ltd, Oxford, UK, forexample, which references are incorporated herein by reference in theirentirety. The obtained antibodies may be polyclonal antibodies ormonoclonal antibodies.

Preferably, at least one species of a modified albumin is isolated andidentified for use in determining a propensity for kidney disease. Theisolated species may be used to generate antibodies for use inimmunoassays. The antibodies may be tagged with an enzymatic,radioactive, fluorescent or chemiluminescent label. The detection methodmay include, but is not limited to radioimmuoassay, immunoradiometricassay, fluorescent immunoassay, enzyme linked immunoassay, and protein Aimmunoassay. The assays may be carried out in the manner described inGoding J W, Monoclonal Antibodies: Principles and Practice. Productionand Application of Monoclonal Antibodies in Cell Biology, Biochemistryand Immunology. 2nd Edition 1986, Academic Press, London, UK; JohnstoneA, Thorpe R, Immunochemistry in Practice, 3rd edition 1996, BlackwellScience Ltd, Oxford, UK; or Price C P, Newman D J (eds.) Principles andPractice of Immunoassay, 2nd Edition, 1997 Stockton Press, New York,N.Y., USA, for example, which references are incorporated herein byreference in their entirety.

It is an object of this invention to provide an article of matter or akit for rapidly and accurately determining the presence or absence ofmodified protein such as modified albumin, in a sample quantitatively ornon-quantitatively as desired. Each component of the kit(s) may beindividually packaged in its own suitable container. The individualcontainer may also be labeled in a manner, which identifies thecontents. Moreover, the individually packaged components may be placedin a larger container capable of holding all desired components.Associated with the kit may be instructions, which explain how to usethe kit. These instructions may be written on or attached to the kit.

The invention is also directed to a method of determining a treatmentagent for renal disease and/or renal complications of a disease,comprising:

(a) administering to a person an agent that is suspected of being ableto treat the disease;

(b) obtaining a urine sample from the person; and

(c) assaying for the modified form of the protein in the sample, whereineither the presence of or lack of presence of a modified form of theprotein in the urine or decreasing amount of the modified form of theprotein over time indicates that the agent is a treatment agent for thedisease. The treatment agent may be a lysosome activating agent that mayact directly or indirectly to activate lysosome, and thereby cause thelysosome to digest post-glomerular filtered proteins, which is a sign ofa healthy kidney.

The process of trafficking of proteins to the lysosomes plays a role inthe mechanism of albuminuria in diabetes. An intracellular molecule thatis involved in trafficking is protein kinase C (PKC). It is contemplatedthat a drug or agent can be formulated that will activate lysosomaltrafficking or inhibit PKC.

Accordingly, in one aspect of the present invention, there is provided alysosome-activating compound for use in reactivating lysosomes orprocesses that direct substrates to the lysosome or products away fromthe lysosome.

In another aspect of the present invention, there is provided acomposition comprising a lysosome-activating compound and a carrier.

In yet another aspect of the invention there is provided a method ofpreventing or treating kidney disease, said method includingadministering an effective amount of a lysosome-activating compound to asubject.

In yet another aspect of the present invention, there is provided amethod of screening a multiplicity of compounds to identify a compoundcapable of activating lysosomes or processes that direct substrates tothe lysosome or products away from the lysosome, said method includingthe steps of:

(a) exposing said compound to a lysosome and assaying said compound forthe ability to activate a lysosome wherein said lysosome when activatedhas a changed activity;

(b) assaying for the ability to restore a cellular process tosubstantially normal levels in kidney tissue, wherein said kidney tissuehas a low lysosome activity; and/or

(c) assaying for the ability to restore tissue turnover to substantiallynormal levels in kidney tissue, wherein said kidney tissue has lowlysosome activity.

Lysosomes may be associated with the breakdown of proteins, particularlyalbumin, in the kidney. In cases of microalbuminuria, substantialamounts of albumin escape lysosomal breakdown possibly due to adeactivated lysosome. Restoration of lysosomal breakdown may restore thebalance in the kidney of cellular processes and tissue turnover.

A lysosome-activating compound may be a compound that acts directly orindirectly on the lysosome. By acting indirectly, the compound may acton a component, which influences the activity of the lysosome.Nevertheless, the outcome results in an activation of the lysosome,thereby providing enhanced protein breakdown.

In another aspect of the present invention, there is provided acomposition comprising a lysosome-activating compound and a carrier.

The composition may be a physiologically acceptable or pharmaceuticallyacceptable composition. However, it will be a composition which allowsfor stable storage of the lysosome activating compound. Where thecomposition is a pharmaceutically acceptable composition, it may besuitable for use in a method of preventing or treating kidney disease.

In yet another aspect of the invention there is provided a method ofpreventing or treating kidney disease, said method includingadministering an effective amount of a lysosome-activating compound to asubject.

As described above, the lysosome-activating compound may act byreactivating the lysosome so that cellular processes and tissue turnoverare restored fully or in part, thereby resulting in the kidney beingrestored partially or fully. In any case, administering a lysosomeactivating compound to an animal having kidney disease may restorelysosome activity fully or in part.

Methods of administering may be oral or parenteral. Oral may includeadministering with tablets, capsules, powders, syrups, etc. Parenteraladministration may include intravenous, intramuscular, subcutaneous orintraperitoneal routes.

The changed activity of the lysosome is preferably a change whichenhances the activity of the lysosome so that albumin breakdown isimproved. The ability to not only activate lysosome but also improvecellular processes and/or tissue turnover is a characteristic of themost desirable lysosome activating compound. Preferably, it is desiredto use the lysosome activating compound to restore kidney function.

In another aspect of the present invention there is provided a methodfor preventing kidney disease in a subject, said method including:

(a) measuring the amount of intact and modified intact albumin contentin a urine sample;

(b) determining a change in the amount of intact albumin in the urinethat has been modified so as to be not detectable by conventional RIAmethods wherein the change is indicative of a propensity for kidneydisease; and

treating the animal for a kidney disease when a change is determined.

The following examples are offered by way of illustration of the presentinvention, and not by way of limitation.

EXAMPLES Example 1 Size Exclusion Chromatography of Human Serum Albumin(HSA)

Normal, healthy volunteers were used to provide urine for analyzing thedistribution of albumin in their urine.

³H[HSA] (Human Serum Albumin) was injected into healthy volunteers andurine and plasma were collected and analyzed by size exclusionchromatography using a G-100 column. The column was eluted with PBS(pH=7.4) at 20 ml/hr at 4° C. The void volume (V_(o)) of the column wasdetermined with blue dextran T2000 and the total volume with tritiatedwater.

Tritium radioactivity was determined in 1 ml aqueous samples with 3 mlscintillant and measured on a Wallac 1410 liquid scintillation counter(Wallac Turku, Finland).

FIG. 2 illustrates the distribution of albumin in urine and in plasma.

Example 2 Albumin Excretion in a Normal Healthy Volunteer and DiabeticPatient

³H[HSA] as used in Example 1 was injected into a normal, healthyvolunteer and a diabetic patient. Samples of urine were collected and³H[HSA] was determined as in Example 1.

The normal, healthy volunteer (FIG. 3) shows the excretion of fragmentsof albumin on a size exclusion chromatography as performed in Example 1.

The diabetic patient (FIG. 4) shows the presence of substantiallyfull-length and fragmented albumin on size exclusion chromatography.However, excretion rates of albumin detectable by these methods were inthe order of 5 μg/min (control) and 1457 μg/min (diabetic).

Example 3 Determination of Intact Albumin, and Intact/Modified Albuminon HPLC.

Urine samples were collected from normal, healthy volunteer,normoalbuminuric diabetic patients and from macroalbuminuric patients.Urine was collected midstream in 50 ml urine specimen containers. Theurine was frozen until further use. Prior to HPLC analysis the urine wascentrifuged at 5000 g.

Samples were analyzed on HPLC using a hydrophobicity column Zorbax 300SB-CB (4.6 mm×150 mm). A 50 μl sample loop was used.

Samples were eluted from the columns using the following conditions.

Solvent A H₂O, 1% trifluoro acetic acid

Solvent B 60% acetonitrile, 0.09% TFA

Solvent A2 99.96>00.00:49.58 min

Pressure 9.014Mpascalls (˜1100 psi)

Solvent B2 0.04>100.0:49.58 min

Pressure 7.154Mpascalls

A wavelength of 214 nm was used.

Example 4 Purification of Modified Albumin for Antibody Production byStandard Techniques

Urine from microalbuminuric patient which had an intact albuminconcentration of 43.5 mg/L as determined by turbitimer (involvingconventional immunochemical assay) was initially filtered through a 30kDa membrane to separate the modified albumin from low molecular weight(<30,000) protein fragments in urine. The material that was retained bythe filter gave a yield of intact albumin of 27.4 mg/L as determined byturbitimer assay. This retained material was then subjected to sizeexclusion chromatography on Sephadex G100. The material collected wasthe peak fraction that coelutes with intact albumin. This material gavea yield of 15.2 ml/L of albumin as determined by the turbitimer method.This material was then subjected to affinity chromatography on an intactalbumin antibody column. This column will only bind albumin that hasconventional epitopes. The yield of material that eluted from the columnwas <6 mg/L (lowest sensitivity of the turbitimer). This is expected asthe immunoreactive albumin would have bound to the affinity column. Theeluate was then subject to reverse phase HPLC chromatography (asdescribed above) to determine the amount of immuno-unreactive albumin inthe sample. A 1452 unit area corresponding to 30.91 mg/L of purifiedmodified albumin was noted as shown in FIG. 5. This purified modifiedalbumin can then be used for antibody production by standard means.

Results

FIG. 5 illustrates a HPLC profile of albumin alone. Essentially a singlepeak which elutes at approximately 39-44 minutes retention time wasobtained.

FIG. 6 illustrates a HPLC profile of plasma showing a distinct albuminpeak at approximately 39-44 minutes as well as other peaks correspondingto other plasma proteins.

FIG. 7 illustrates a HPLC profile of a normal, healthy volunteer showingno albumin peak in the urine sample. This individual breaks down thealbumin excreted into the urine possibly via an active lysosome.Substantial fragmented products were evident showing prominence of somespecies, particularly of a species at approximately less than 14.5minutes retention time.

When urine from a normoalbuminuric diabetic patient (with an albuminexcretion rate of 8.07 μg/min, as measured by RIA) is analyzed (FIG. 8),small amounts of modified albumin eluting at approximately 39-44 minutesretention time is evident. Whereas conventional test indicates thepresence of <6 mg/l of albumin in the urine sample, the method of theinvention showed that the true albumin content in the urine sample was26.7 mg/l. Treatment for the disease should have begun on thisindividual. Albumin by-products or fragmented albumin is present as inthe normal, healthy volunteer.

Another urine sample from normoalbuminuric diabetic patient (withalbumin excretion rate of 17.04 μg/min) was analyzed (FIG. 9). RIA testsshow albumin excreted in the urine for this patient. However, on HPLC(FIG. 9) an albumin or modified albumin peak is evident at approximately39-44 minutes retention time. Whereas conventional test indicates thepresence of <6 mg/l of albumin in the urine sample, the method of theinvention showed that the true albumin content in the urine sample was81.3 mg/l. Treatment for the disease should have begun on thisindividual. This peak begins to show a multiple peaked appearance. Asmaller peak corresponding to intact albumin shows that modified albuminmay represent the peak at 39-44 minutes. The presence of this albuminpeak compared with the profile of a normal, healthy volunteer having noalbumin peak shows a change in the detectable levels of the amount ofintact/modified albumin. This may signal a propensity for a kidneydisease.

A further urine sample from a normoalbuminuric diabetic patient (with analbumin excretion rate of 4.37 μg/min) was analyzed, and the HPLCprofile is illustrated in FIG. 10. Again, modified albumin was detectedat approximately 39-44 minutes retention time showing multiple peaks.This patient again did register normal albumin by RIA. Whereasconventional test indicates the presence of <6 mg/l of albumin in theurine sample, the method of the invention showed that the true albumincontent in the urine sample was 491 mg/l. Treatment for the diseaseshould have begun on this individual. It is clear that modified albuminassessment is necessary to identify these changes. This patient would bedetermined to have a propensity for kidney disease. As kidney diseaseprogresses, the modified albumin peak will continue to increase.

This is shown in FIG. 11 where a urine sample of a macroalbuminuricpatient was analyzed. A quite significant albumin peak at approximately39-44 minutes retention time showing multiple peaks was evident. Thepatient's albumin content was 1796 mg/l. Treatment for this individualis in progress.

The method of the invention results in early detection of a propensityfor a renal disease as illustrated by the longitudinal studies in FIGS.12-14. FIGS. 12-14 show situations in which the ACE inhibitor treatmentfor diabetes was begun later than it should have had the modifiedalbumin detection method of the invention been used. Detecting modifiedprotein using the method according to the invention is a more effectivemethod for predicting the onset of a renal disease than usingconventional RIA.

All of the references cited herein are incorporated by reference intheir entirety.

Finally, it is to be understood that various other modifications and/oralterations may be made without departing from the spirit of the presentinvention as outlined herein.

What is claimed is:
 1. A method for measuring albumin content in a urinesample, comprising assaying the urine sample for albumin by conventionalassay and assaying for intact modified albumin.
 2. The method accordingto claim 1, wherein said conventional assay comprises an antibody methodor fractionating said sample via chromatography, electrophoresis orsedimentation to test for native and/or intact modified albumin.
 3. Themethod according to claim 1, wherein assaying for intact modifiedalbumin comprises fractionating said sample via chromatography,electrophoresis or sedimentation, or use of a specific albumin dyemethod to test for native and/or intact modified albumin in said urinesample.
 4. A method for detecting protein content in a urine sample bynon-antibody assay, comprising detecting a sum of native protein andintact modified protein in the urine sample.
 5. The method according toclaim 4, wherein said non-antibody assay comprises fractionating theprotein in said sample via chromatography, electrophoresis orsedimentation to test for the presence of native and/or intact modifiedprotein.
 6. A method for diagnosing kidney disease or renalcomplications of disease prior to the onset of kidney degeneration in apatient by detecting protein content in a urine sample obtained from thepatient, comprising detecting intact modified protein and native proteinamount in the sample by non-antibody assay; and correlating the presenceof native and/or intact modified protein in the urine to the presence ofkidney disease or renal complications of disease.
 7. The methodaccording to claim 6, wherein said non-antibody assay comprisesfractionating the protein in said sample via chromatography,electrophoresis or sedimentation to test for the presence of nativeprotein and/or intact modified protein.
 8. The method according to claim6, wherein said protein is albumin.
 9. The method according to claim 6,wherein the disease comprises nephropathy, diabetes insipidus, diabetestype I, diabetes II, renal disease glomerulonephritis, bacterial orviral glomerulonephritides, IgA nephropathy, Henoch-Schönlein Purpura,membranoproliferative glomerulonephritis, membranous nephropathy,Sjögren's syndrome, nephrotic syndrome minimal change disease, focalglomerulosclerosis and related disorders, acute renal failure, acutetubulointerstitial nephritis, pyelonephritis, GU tract inflammatorydisease, Pre-clampsia, renal graft rejection, leprosy, refluxnephropathy, nephrolithiasis, genetic renal disease, medullary cystic,medullar sponge, polycystic kidney disease, autosomal dominantpolycystic kidney disease, autosomal recessive polycystic kidneydisease, tuborous sclerosis, von Hippel-Lindau disease, familialthin-glomerular basement membrane disease, collagen III glomerulopathy,fibronectin glomerulopathy, Alport's syndrome, Fabry's disease,Nail-Patella Syndrome, congenital urologic anomalies, monoclonalgammopathies, multiple myeloma, amyloidosis and related disorders,febrile illness, familial Mediterranean fever, HIV infection—AIDS,inflammatory disease, systemic vasculitides, polyarteritis nodosa,Wegener's granulomatosis, polyarteritis, necrotizing and crecenticglomerulonephritis, polymyositis-dermatomyositis, pancreatitis,rheumatoid arthritis, systemic lupus erythematosus, gout, blooddisorders, sickle cell disease, thrombotic thrombocytopenia purpura,hemolytic-uremic syndrome, acute corticol necrosis, renalthromboembolism, trauma and surgery, extensive injury, burns, abdominaland vascular surgery, induction of anesthesia, side effect of use ofdrugs or drug abuse, malignant disease, adenocarcinoma, melanoma,lymphoreticular, multiple myeloma, circulatory disease myocardialinfarction, cardiac failure, peripheral vascular disease, hypertension,coronary heart disease, non-atherosclerotic cardiovascular disease,atherosclerotic cardiovascular disease, skin disease, soriasis, systemicsclerosis, respiratory disease, COPD, obstructive sleep apnoea, hypoiaat high altitude or erdocrine disease, acromegaly, diabetes mellitus, ordiabetes insipidus.
 10. The method according to claim 6, wherein theprotein comprises albumin, globulin, α₁-globulin, α₂-globulin,β-globulin, γ-globulin, euglobulin, pseudoglobulin I or II, fibrinogen,α₁-acid glycoprotein, α₁-glycoprotein, α₁-lipoprotein, ceruloplasmin, α₂19S glycoprotein, β₁ transferrin, β₁ lipoprotein, immunoglobulins A, E,G, or M, horseradish peroxidase, lactate dehydrogenase, glucose oxidase,myoglobin, lysozyme, protein hormone, growth hormone, insulin orparathyroid hormone.
 11. The method according to claim 7, wherein theprotein is albumin.
 12. The method according to claim 7, wherein saidnon-antibody assay comprises partition chromatography, adsorptionchromatography, paper chromatography, thin-layer chromatography,gas-liquid chromatography, gel chromatography, ion-exchangechromatography, affinity chromatography, or hydrophobic interactionchromatography, moving-boundary electrophoresis, zone electrophoresis,or isoelectric focusing.
 13. The method according to claim 12, whereinthe non-antibody assay is hydrophobic interaction chromatography carriedout in a high pressure liquid chromatography (HPLC) apparatus.
 14. Amethod for detecting renal disease caused by diabetes comprising: i)collecting a series of urine samples from a patient over a period oftime, ii) subjecting each urine sample in the series of samples toanalysis by chromatography, electrophoresis, sedimentation, or antibodyassay, iii) detecting an increase in the sum of native protein and/orintact modified protein in the samples over the period of time, whereinan increase in the amount of the intact modified protein and nativeprotein or intact modified protein is indicative of the presence of therenal disease.
 15. A method for detecting renal disease caused bydiabetes by the steps comprising: i) collecting a urine sample from aperson, ii) subjecting the urine sample to analysis by chromatography,electrophoresis, sedimentation apparatus, or antibody means; and iii)detecting the presence of intact modified albumin in the sample, whereinthe presence of the intact modified albumin in indicative of thepresence of the renal disease.
 16. The method according to claim 14,wherein the modified protein is detected by an antibody that is specificfor native and/or intact modified forms of the protein.
 17. The methodaccording to claim 16, wherein the antibody is specific for the modifiedprotein.
 18. The method according to claim 14, wherein the antibody isattached to an enzymatic, radioactive, fluorescent or chemiluminescentlabel, wherein the detecting step comprises radioimmunoassay,immunoradiometric assay, fluorescent immunoassay, enzyme linkedimmunoassay, or protein A immunoassay.
 19. The method according to claim14, wherein an early stage of the disease prior to the onset of kidneydegeneration is diagnosed when the intact modified protein is present inthe urine in increasing amounts over time.
 20. An article of manufacturefor diagnosing early stage of a disease in which intact modified proteinfound in urine is an indicator of the disease, comprising: (a) acontainer comprising a labeled antibody specific for said modifiedprotein; (b) a container comprising reagents for developing an antibodyreaction; and (c) instructions on how to use components (a) and (b) tocarry out the diagnosis.
 21. The method according to claim 4 wherein theprotein is albumin and the non-antibody assay comprises use of analbumin specific dye to test for native and intact modified albumin. 22.The method according to claim 6, wherein the protein is globulin. 23.The method of claim 4 wherein the protein is selected from albumin,transferrin and IgG.
 24. The method according to claim 14 wherein theprotein is albumin and the non-antibody assay comprises use of analbumin specific dye to test for native and intact modified albumin.